As prior arts concerning the process for preparing cyclosporin A, methods using fermentation by Cylindrocarpon lucidum and Tolypocladium inflatum are disclosed in GB Patent 1,491,509 and U.S. Pat. No. 4,117,118, and U.S. Pat. No. 4,215,199. Also, various methods to recover cyclosporin A from the culture fluid are disclosed in these patents.
In the above-mentioned patents, a process comprising the steps of extracting mycellium isolated from the culture fluid with a solvent, and then recovering the product by successively using chromatography media such as sephadex, aluminum oxide and silica gel has been used as a process for recovering cyclosporin A from the culture fluid. In accordance with these processes, solvent is added to mycellium as isolated solid obtained by filtering the culture fluid; the mixture is extracted by solvent by means of centrifugal extracter; the resultant extract is pretreated with water or hexane or the like; and then the product is purified through chromatography media such as sephadex, aluminum oxide and silica gel, etc. to obtain cyclosporin A. However, the procedure is complicated because two or three types of chromatography media including sephadex should be used after extracting the mycellium as isolated solid obtained by filtering the culture fluid with solvent. Besides, the processes may cause problems of process economics and environmental pollution because each eluting solvent shoud be used for each chromatography procedure.
Thus, in order to overcome the complexity of the processes and lighten the economic burden, various methods for purifying cyclosporin A have been studied.
For example, according to the teachings of U.S. Pat. No. 5,256,547 and European Patent 507,968 A1, mycellium as isolated solid obtained by filtering the culture fluid is dried and then extracted with a solvent to obtain crude cyclosporin A; the product is pretreated without washing with water or the like, and purified by means of chromatography using aluminum oxide and silica gel avoiding the use of expensive sephadex to obtain cyclosporin A. In addition, according to the teachings of U.S. Pat. No. 5,156,960, the culture fluid is extracted by directly adding solvent, the extract is transferred to a solvent having higher solubility and dried over a drying agent such as anhydrous magnesium sulfate, and the resultant product is purified by chromatography through sephadex and silica gel etc. to obtain cyclosporin A.
Although the art described in U.S. Pat. No. 5,256,547 has advantages in that content of pigment has been reduced by virtue of the solvent extraction and the process has been simplified in the course of solvent pretreatment and chromatography, it also has disadvantage in that an extra process for drying the mycellium should be performed. On the other hand, the art described in U.S. Pat. No. 5,156,960 has an advantage in that solvent extraction is carried out directly from the culture fluid by simply adding a solvent instead of solvent extraction from mycellium separated from the culture fluid after filtration, and the processes through chromatography media are performed without pretreatment with solvent. However, there should be an additional process for drying over expensive anhydrous magnesium sulfate because a significant amount of moisture is admixed in the transferring solvent owing to the curtailment of the process, and two chromatography media of sephadex and silica gel are used so that it still involves problems of complexity and economic disadvantage so as to be industrialized.